PROJECT SUMMARY Evolution of antibiotic resistance is a global public health problem. How evolution renders antibiotic molecules ineffective by altering antibiotic targets is an interesting phenomenon from both clinical and basic science perspectives. In pathogenic bacteria, there is only a handful of drug target enzymes such as DNA gyrases, RNA polymerases, fatty acid synthetases, and enzymes involved in folic acid synthesis. Therefore, a mechanistic understanding of resistance-conferring mutations in these enzymes is clinically critical for designing new drugs or drug variants that can inhibit resistant bacteria. In this project, we propose to study evolution of the Escherichia coli dihydrofolate reductase (DHFR) enzyme and map epistatic interactions between DHFR mutations. DHFR is a ubiquitous enzyme with an essential role in the folic acid synthesis pathway and is used as a drug target in antibacterial, anticancer, and antimalarial therapies. In bacteria, an antibiotic named trimethoprim competitively binds to DHFR and blocks its catalytic activity. Therefore, DHFR mutations that either confer resistance or compensate for reduced catalytic activity of resistant DHFR mutants are selected for bacterial survival. We will use laboratory evolution experiments to identify functional DHFR mutations and reproducible genetic trajectories leading to elevated trimethoprim resistance. We will characterize these mutations by using in vitro biochemical assays and deep-sequencing based fitness measurements for calculating epistatic interactions between DHFR mutations. We will use molecular dynamics along with other computational tools and nuclear magnetic resonance (NMR) spectroscopy to reveal structural changes responsible for resistance and epistatic interactions. The combination of these approaches presents a unique opportunity to quantitatively evaluate evolutionary paths leading to trimethoprim resistance and create a discovery pipeline for studying protein evolution. By creating a deeper understanding for the evolutionary dynamics of an important drug target enzyme, our proposal will develop experimental and computational tools for studying protein evolution with the ultimate goal of improving human health. Indeed, our preliminary analyses suggest that we will be able to design and test novel trimethoprim derivatives that can selectively inhibit DHFR mutants that carry the L28R replacement, a common and synergistic DHFR mutation. We propose to synthesize trimethoprim-Dihydrofolate hybrid molecules that will possess the salient structural features of both DHF and trimethoprim molecules selectively inhibit DHFR mutants with the L28R replacement. We will evolve pan sensitive E. coli strains in the morbidostat in order to quantify the efficacy of the mutant specific trimethoprim derivatives in impeding resistance evolution and accordingly develop new strategies for better use of it.